Mycobacterium tuberculosis can be rapidly diagnosed through nucleic acid amplification techniques by analyzing the variations in the associated gene sequences. In the present study, a locked nucleic acid (LNA) probe-based real-time PCR assay was developed to identify the mutations in the rpoB gene associated with rifampin (RFP) resistance in M. tuberculosis. Six LNA probes with the discrimination capability of one-base mismatch were designed to monitor the 23 most frequent rpoB mutations. The target mutations were identified using the probes in a “probe dropout” manner (quantification cycle = 0); thus, the proposed technique exhibited superiority in mutation detection. The LNA probe-based real-time PCR assay was developed in a two-tube format with three LNA probes and one internal amplification control probe in each tube. The assay showed excellent specificity to M. tuberculosis with or without RFP resistance by evaluating 12 strains of common non-tuberculosis mycobacteria. The limit of detection of M. tuberculosis was 10 genomic equivalents (GE)/reaction by further introducing a nested PCR method. In a blind validation of 154 clinical mycobacterium isolates, 142/142 (100%) were correctly detected through the assay. Of these isolates, 88/88 (100%) were determined as RFP susceptible and 52/54 (96.3%) were characterized as RFP resistant. Two unrecognized RFP-resistant strains were sequenced and were found to contain mutations outside the range of the 23 mutation targets. In conclusion, this study established a sensitive, accurate, and low-cost LNA probe-based assay suitable for a four-multiplexing real-time PCR instrument. The proposed method can be used to diagnose RFP-resistant tuberculosis in clinical laboratories.Drug-resistant  Mycobacterium tuberculosis has been considered as one of the most critical public health issues. The early diagnosis of drug-resistant tuberculosis via a rapid and reliable method can facilitate the administration of effective anti-tuberculosis drugs to patients and prevent the further spread of drug-resistant strains  and a BHQ at the 3′-end. All of the probes and primers were synthesized by Huirui .The real-time PCR primers and probes used in the study are shown in Bacillus globigii (ATCC 9372) were also used for the internal amplification control (IAC) and were amplified with M. tuberculosis in one real-time PCR to detect PCR inhibitors. The oligonucleotide sequences of the primers and a Taqman probe were designed using Primer Express 3.0  to target a conserved segment of BgSP in B. globigii (GenBank accession no. L27989). The Taqman probe was labeled with carboxy-X-rhodamine (ROX) to be distinguished from the other dyes.The specific primers and probes of rpoB , reverse primer of rpoB , detecting LNA-probe (240 nM), and 2.5 μl of plasmid DNA template (3.0 × 105 copies/μl). The final volume was adjusted with distilled water. The reaction was run under the following conditions: 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 65°C for 30 s, and a cooling down step at 37°C for 1 min.Plasmid DNA templates, including the wild-type template and the corresponding mutant templates, were used to determine the specificity of each LNA probes through a monoplex-probe real-time PCR amplification. The monoplex-probe real-time PCR was performed in a final reaction volume of 25 μl containing 2× LightCycler 480-probe master mix (Roche Diagnostics), forward primer of rpoB , primers for BgSP , a mixture of probes , or a mixture , 2.5 μl of the purified DNA of B. globigii (1000 GE/μl), and 2.5 μl of the genomic DNA extracted from mycobacteria.Multiplex-probe real-time PCR was run in a two-tube formation (Tube A and Tube B), with three differently labeled LNA-probes and an IAC-probe in each tube. A final reaction volume of 30 μl was used in each tube containing 2× LightCycler 480-probe master mix, primers for Real-time PCR was conducted using a LightCycler 480 II instrument. The conditions of PCR amplification were as follows: initial activation at 95°C for 5 min, followed by 45 cycles of denaturation at 95°C for 15 s, annealing at 65°C for 30 s, and a cooling down step at 37°C for 1 min. Four filter combinations (excitation spectrum–emission spectrum) were chosen and set as follows: FAM channel (465 nm–510 nm), HEX channel (498 nm–580 nm), Rox channel (533 nm–610 nm), and Cy5 channel (618 nm–660 nm). The fluorescence in the four channels was determined simultaneously during the annealing step. Before fluorescence was analyzed, color compensation was established in accordance with the manufacturer’s instructions. The quantification cycle (Cq) was obtained by using the LightCycler software program (version 4.0) with the second derivative algorithms.M. tuberculosis was determined by positive signals from at least four LNA probes; (2) RFP-susceptible M. tuberculosis was determined by positive signals from all of the six LNA probes; (3) RFP-resistant M. tuberculosis was determined by one or two absent probe signals. (4) M. tuberculosis was precluded if signals were absent in three or more probes. (5) Diagnoses were based on a positive signal from the IAC probe (34 < Cq < 40); otherwise, the test was considered invalid.The real-time PCR assay results are described as follows: (1) M. tuberculosis H37Rv was prepared in serial dilutions from 4,000,000 GE/μl to 4 GE/μl to evaluate the assay sensitivity. Then, 2.5 μl of the diluted DNA was used as the template to determine sensitivity for the multiple-probe-based real-time PCR. The templates are tested in triplets at least, as described above. To achieve an improved sensitivity, the nested PCR was introduced into the assay. The first round of the nested PCR was performed in a 20 μl reaction volume containing 2× Light Cycler 480-probe master mix, 250 nM of each primer , 3 μl of DNA, and distilled water. The reaction was run under the following conditions: 95°C for 5 min, followed by 30 cycles of 10 s at 95°C, 15 s at 60°C, and another 15 s at 72°C. The product from the first round of PCR was diluted 100 times with distilled water and used as the template for the second round of real-time PCR as mentioned above.The genomic DNA of rpoB mutations associated with RFP resistance. Of the templates, 15 were from the genomic DNA of RFP-resistant strains of M. tuberculosis among the sequenced clinical isolates; the other 8 DNA templates were from the plasmid DNA prepared in the study. All the DNA templates were diluted to the concentration ranged from 105 to 106 GE/tube or copies/tube.DNA samples with 23 different RRDR sequences were prepared to determine the assay capability to discriminate M. tuberculosis and drug-resistant M. tuberculosis were also prepared and tested as the control.12 NTM strains were collected and used to confirm the detecting specificity of the proposed assay. Genomic DNA samples were extracted from the NTMs as the templates (approximately 0.1 ng/μl) of the LNA-probe based real-time PCR. DNA sequences from drug-susceptible rpoB sequence, were used to evaluate performance of the assay. Each DNA sample was diluted to the concentration of 105 or 106 GE/tube as the template of the real-time PCR. The DNA samples were coded independently and randomly and tested by the assay in a blind test way.A total of 154 bacteria samples, including 12 NTM strains and 142 clinical isolates with well-defined information on drug susceptibility and rpoB gene of M. tuberculosis. The probes were assigned to monitor the RRDR region (from codon 507 to codon 533 of rpoB). The LNA probes could identify these single-base mutations in a manner of “probe dropout” (Cq = 0); in this way, the probe could not combine with the mutant target; as a result, negative fluorescence signals were produced during amplification. The six LNA probes were allocated in two tubes to consist of a two-tube form real-time PCR assay. Each tube contained three LNA probes labeled with different dyes. The two reactions were performed simultaneously and shared the same real-time PCR components except the included probes to detect drug-resistant M. tuberculosis. Probe allocation was determined by comparing different probe combinations  was developed to monitor PCR inhibitors in each tube. The IAC system consists of specific primers and a Taqman probe labeled with Rox, which were targeted at a conserved segment in BgSP of B. globigii. Considering the amplification competition between rpoB and BgSP, we pre-added approximately 2500 GE/tube B. globigii to the reaction mixture to generate a sufficient IAC signal and significantly reduce the interference with rpoB amplification.An internal amplification control (IAC) based on rpoB sequences with single-base mutations were prepared as template. The results showed that the 23 types of mutant genotypes could be clearly differentiated from the wild targets on the basis of the amplification curves; these mutant genotypes were detected through “probe dropout”. The Cq values of the mutant sequences were determined as negative (Cq = 0) by the second derivative algorithms described in the Materials and methods. The results showed the strong capability of the LNA probe to identify single-base mutations.Before the assay with multiple probes was established, the capability of each LNA probe to discriminate single-base mutations was initially confirmed in monoplex-probe real-time PCR experiments. Plasmid DNA samples, including the wild-type strains and 23 common types of 6 copies/tube) did not significantly vary, although the six probes were aimed at different regions and emitted signals in different fluorescent channels  to determine the sensitivity of the assay. The results indicated that the limit of detection (LOD) could reach 100 GE/tube with an average Cq value of 37.72 . The curve displayed good linearity (R2 = 0.987) for M. tuberculosis at a range of 1.0 × 102 GE/tube to 1.0 × 106 GE/tube. The quantification equation was expressed by y = −3.525x + 52.270. The equation revealed an amplification efficiency of 92.2% for the assay; thus, the multiple-probe PCR was strongly reliable.The quantification curve was also established to quantitatively detect rculosis . The x-arpoB mutations associated with RFP resistance was evaluated to detect RFP-resistant M. tuberculosis. As shown in rpoB genotypes with single-base mutations could be correctly detected through the assay. The absence of any single-base mutation in the DNA caused the “dropout” of the corresponding LNA probe during amplification (Cq = 0); by contrast, the other LNA probes worked well in the reaction. Our results further indicated that the RFP-resistant M. tuberculosis can be identified on the basis of the “probe dropout” instead of ΔCq values that is used in most other NAAT assays [rpoB mutations associated with RFP resistance are mainly single- or double-codon mutations, and triple or more codon mutations have been rarely reported. Therefore, RFP-resistant M. tuberculosis could be determined as positive if one- or two-probe dropout from the target DNA.The capability of the assay to discriminate T assays , 15. PreM. tuberculosis was confirmed by evaluating the DNA samples extracted from 12 NTM strains (M. tuberculosis did not have probe dropouts (RFP-susceptible M. tuberculosis) or did not have more than two dropouts (RFP-resistant M. tuberculosis). However, three or more LNA probe dropouts occurred when the 12 NTM DNA samples were evaluated . Furthermore, of the 142 strains identified as M. tuberculosis, 88/88 (100%) RFP-susceptible M. tuberculosis strains were accurately detected through the assay. In addition, 52/54 (96.3%) of the RFP-resistant M. tuberculosis strains were correctly determined. Of the total samples, only two RFP-resistant strains were falsely diagnosed as RFP-susceptible M. tuberculosis by the assay. Corresponding DNA Sequences of the RRDR was analyzed in the two discordant samples. An rpoB mutation at codon 510 (CAG-CAC) and a mutation codon 522 (TCG-ATG) were then found in the two RFP-resistant samples. However, both mutations were related to low level of resistance to RFP [M. tuberculosis and RFP-resistant M. tuberculosis.A set of 154 clinical isolates with well-defined information on drug susceptibility and lts show , 12/12  to detect M. tuberculosis and RFP-resistant M. tuberculosis.In the study, we developed a LNA probe-based assay to determine the genotype of The proposed assay utilized six well-designed LNA probes as the genotype detector of RRDR. As a result, all the 23 common mutations were highly distinguished by the LNA probes in a manner of “probe dropout”, which could be easily identified through the amplification curves (Cq = 0), without complex determinations or additional Tm analyses. There are several real-time PCR assays developed for the detection of drug-resistance associated mutations by using other kinds of probes, such as the molecular beacon-based Xpert MTB/RFP assay , HoweverM. tuberculosis.The assay contains seven different probes, including six LNA probes and one IAC Taqman probe. Considering the availability of the real-time PCR instrument and easy application of the assay, we developed a real-time PCR assay in a two-tube form instead of one tube. The design requires only a real-time PCR instrument with four fluorescent detecting channels, such as LightCycler 480 II, ABI 7500, SmartCycler, and CFX 96 real-time PCR detecting system. However, the assay could be improved to a single-tube format to exhibit convenience; thus, the assay could be applicable to common instruments. Furthermore, the assay can be transferred to an automatic diagnostic platform with the capability of “sample-in answer out,” similar to the Cepheid GeneXpert system , to achirpoB, and these probes have been validated with excellent specificity to detect the common mutations in the RRDR. Moreover, the LNA probe-based real-time assay developed in the study shows sufficient specificity and sensitivity to detect M. tuberculosis and drug-resistant M. tuberculosis, which can become a powerful alternative tool to rapidly diagnose drug-resistant tuberculosis. The assay could also be used to detect the mutations involved in the resistance to other anti-tuberculosis drugs, such as isoniazid, ethambutol, and fluoroquinolone, through designing corresponding specific LNA probes, in future studies.In summary, six LNA probes were designed to detect drug resistance-associated mutations in S1 Fig(TIF)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.


As a common toxic metal, lead has significant neurotoxicity to brain development. Long non-coding RNAs (lncRNAs) function in multiple biological processes. However, whether lncRNAs are involved in lead-induced neurotoxicity remains unclear. Uc.173 is a lncRNA from a transcribed ultra-conservative region (T-UCR) of human, mouse and rat genomes. We established a lead-induced nerve injury mouse model. It showed the levels of Uc.173 decreased significantly in hippocampus tissue and serum of the model. We further tested the expression of Uc.173 in serum of lead-exposed children, which also showed a tendency to decrease. To explore the effects of Uc.173 on lead-induced nerve injury, we overexpressed Uc.173 in an N2a mouse nerve cell line and found Uc.173 had an inhibitory effect on lead-induced apoptosis of N2a. To investigate the molecular mechanisms of Uc.173 in apoptosis associated with lead-induced nerve injury, we predicted the target microRNAs of Uc.173 by using miRanda, TargetScan and RegRNA. After performing quantitative real-time PCR and bioinformatics analysis, we showed Uc.173 might inter-regulate with miR-291a-3p in lead-induced apoptosis and regulate apoptosis-associated genes. Our study suggests Uc.173 significantly inhibits the apoptosis of nerve cells, which may be mediated by inter-regulation with miRNAs in lead-induced nerve injury. Lead, one of the main heavy metals, is widely distributed in the environment. Lead exposure in the living environment harms human health; in particular, prevalence of lead poisoning is much higher in children than in adults. It has been suggested that lead can act as a kind of poison with strong neural developmental toxicity . Even loLncRNAs are a form of RNA whose length is more than 200 nucleotides. They themselves do not encode proteins but participate in the regulation of gene expression and various biological processes . LncRNAsThere are 481 ultra-conservative regions (UCRs), which share 100% identity among human, mouse and rat genomes . The funIn our previous study, we treated PC12 cells  with lead acetate and made lncRNA chip and RT-qPCR (Reverse Transcript-quantitative PCR) analysis. We found Uc.173 was downregulated significantly among the differentially-expressed genes. To investigate the role of Uc.173 in lead-induced nerve injury, we tested the changes of the expression of Uc.173 in brain tissue, serum of lead-exposed mouse as well as in the serum of lead-exposed humans. We used N2a mouse nerve cells to further explore the effects of Uc.173 on neuronal apoptosis induced by lead. Our results suggest Uc.173 plays an important role in apoptotic inhibition in lead-induced nerve injury.To test the changes of expression of Uc.173 during brain injury induced by lead, we established a C57bl/6 mouse model of lead-induced brain injury. We used inductively coupled plasma mass spectrometry (ICP-MS) to monitor the blood lead concentration in mice. Compared with controls, the blood lead concentration increased slightly in Week 1 and Week 2 of the lead treatment while it increased significantly in Week 5 Figure . The reaWe used RT-qPCR to test the expression of Uc.173 in the hippocampus tissues and serum of lead-exposed mice and in the serum of lead-exposed populations. Each group included ten mice. The animal model was successfully established after five weeks treatment of lead. Then we tested the expression of Uc.173 in mouse hippocampus tissues Figure  and seruTo study the effects of lead exposure on the expression of Uc.173 in nerve cells, we treated mouse neuroblastoma N2a cells with 0.1 um/L lead acetate and observed the changes of Uc.173 expression at various treatment time points. We tested the expression of Uc.173 in N2a cells at five time points  after the treatment and found with the extension of lead-exposure time, the expression of Uc.173 in N2a cells decreased gradually. Compared with 0 h, the expression of Uc.173 decreased by 2.4-fold in 48 h  assay to further verify the anti-apoptotic function of Uc.173. TUNEL assay We then tested the expression of caspase 3 and caspase 9, key members in the apoptotic pathway, by assessing protein levels with a western blot. We tested the control group, the lead-treatment alone group and the Uc.173-overexpressed plus lead-treatment group, respectively. As shown in Figure PI3K, NIK and Cn) or a DNA-damage repair-associated target gene (RAD23B). These target genes are all key regulatory factors in the apoptotic pathway. Among these four miRNAs, only miR-291a-3p was upregulated in the lead-treatment alone group, in which the expression of Uc.173 was actually downregulated. Based on the bioinformatics analysis and the prediction of target genes, we propose that Uc.173 likely inhibits apoptosis through the mediation of miR-291a-3p, which needs further studies to verify.Studies have shown that there is inter-regulation between T-UCR and miRNA. T-UCR can play a regulatory function through the mediation of miRNA . To idenLead is an important heavy metal in the environment. Exposure to lead threatens human health and can influence the intellectual development of children significantly, which mainly presents as learning and memory disorders . DiagnosThe majority of lncRNAs are transcribed by RNA polymerase II. The RNA molecules do not encode proteins due to the lack of open reading frames. LncRNAs are a kind of evolutionarily-conserved RNA molecule whose expression is dynamic and varies with the environment. Compared with protein-coding genes, lncRNAs have tissue and developmental specificity . LncRNAsStudies indicate that lead can induce the apoptosis of nerve cells in the hippocampus and cerebral cortex of developing and mature rats . Cell apCell apoptosis is regulated by a set of genes and is accompanied by changes in the expression of associated proteins , so we fCalin et al.  found thIn conclusion, Uc.173, a T-UCR, plays an important role in lead-induced neuronal apoptosis. Our study reveals a new mechanism of lead-induced nerve injury.n = 30) and lead acetate-treatment group . Each cage fed five mice. The mice in the lead acetate-treatment group were fed with drinking water containing 9.6 mmol/L lead acetate. The mice in the two groups were sacrificed after 1, 2 and 5 weeks treatment of lead acetate (ten mice/group/time point). All processes involving experimental animals were approved by the Animal Care and Use Committee of Guangzhou Medical University. The whole blood of the mice was collected and blood lead concentration was tested by Agilent 7700C ICP-MS . Pathological examination by HE staining was performed on sacrificed mice in the two groups.A batch of 60 ablactating C57bl/6 mice (three weeks old) with a weight range of 10-12 g were purchased from the Medical Animal Experimental Center of Guangdong, China and fed in the specific pathogen free (SPF) animal room of Laboratory Animal Center of Guangzhou Medical University, China. The temperature was 20-26°C and the relative humidity was 40%-70%. The light/dark cycle was 12/12 hours. After a three-day adaptation, the C57bl/6 mice were randomly divided into control group  and with high blood lead concentration (more than 80 ng/L) were paired up for the study.The cellular model was constructed by treating mouse N2a nerve cells with 0.1 μm/L lead acetate solution for 48 hours.The total RNAs (including lncRNAs) of mouse hippocampus tissue were extracted using TRIzol Reagent . The hippocampus tissue was placed in a 1.5 ml centrifuge tube containing 300 μl TRIzol reagent and ground to a paste using an electric micro-tissue homogenizer. TRIzol (700 μl) was added and the grinding continued. After this, the homogenate samples were placed at room temperature for 5 minutes to completely separate the DNA-protein complex. The extraction of total RNAs was performed by following the manufacturer's protocol. The quality and concentration of extracted RNAs were measured by a NanoDrop1000 Spectrophotometer . The total RNAs in mouse serum and human serum were extracted using TRIzol LS (Invitrogen). TRIzol LS Reagent  was added into 100 μl serum and fully mixed. After placing at room temperature for 5 minutes, the total RNAs were extracted according to the TRIzol LS manual.−ΔCt, and in the cellular model it was 2−ΔΔCt.A Prime-Script RT reagent Kit  was used to reverse transcribe RNAs to cDNAs. PCR was performed using SYBR Premix Ex Taq (TaKaRa). The primers of internal control genes and specific primers are shown in Extraction of genomic DNA, culture of receptor bacteria and preparation of competent cells were performed before the construction of the vector. The construction of the vector included the amplification of target fragments, linkage of amplified fragments with pGEM-T Easy-Uc.173  vectors and transfection of competent cells. Transformation: 200 μL of competent cell suspension, previously stored at −80°C, was thawed in an ice bath. Ligation products were added, mixed gently and placed on ice for 30 minutes. The cells were heat shocked for 80 seconds in a water bath at 42°C and immediately placed on ice for 3-5 minutes. One milliliter LB liquid medium (Not containing ampicillin (Amp)), was added to the tube, mixed and incubated for 1 hour at 37°C. One hundred micro liters of bacterial solution was spread onto the screen plate containing Amp and incubated at 37°C for 16-24 hours. Positive clones were analyzed using electrophoresis and then sequenced and transcribed to RNA. The transfection system contained: 12 μl EndoFectin-Lenti  1 μg Uc.173, 200 μl Dulbecco minimum essential medium (DMEM) without serum. Twenty-four hours after transfection, PCR was performed to evaluate transfection efficiency.g, 4°C, 5 min). Approximately 1-5 × 105 cells were collected and resuspended in 100 μl 1×Binding Buffer (KeyGen Biotech). Annex in V-FITC (5 μl KeyGen Biotech) and 5 μl PI Staining Solution (KeyGen Biotech) were added and mixed gently. The reaction lasted for 10 minutes under room-temperature in a light-proof environment. Four hundred micro liters of 1×Binding Buffer was added and mixed gently. FCM was performed within one hour using a flow cytometer . Annexin V-FITC+PI- quadrant represented early apoptotic cells; Annexin V-FITC+PI- quadrant represented early apoptotic cells (LR); Annexin V-FITC-PI+ quadrant represented necrotic cells (UL); Annexin V-FITC+PI+ quadrant represented late apoptotic and necrotic cells (UR); Annexin V-FITC-PI- quadrant represented live cells (LL). Finally, the ratio of apoptotic cells accounting for total cells was calculated.Annexin V-Fluorescein Isothiocyanate Apoptosis Detection Kit  was used to detect cell apoptosis. The cells were digested by non-ethylene diamine tetra acetic acid (EDTA)-containing trypsin 48 hours after transfection and the treatment of lead, and then centrifuged  was used to detect apoptosis. The cells were adherently cultured on a Lab-Tek slide. After treatment with lead acetate, the slide was washed twice with PBS. The sample was fixed using 4% polyformaldehyde at room temperature for 20 minutes and washed twice (5 minutes each time) with PBS. The prepared TUNEL reaction mixture was dropped onto the cover slip and covered by a cover glass. The section was incubated at 37°C for 60 minutes and washed in PBS for three times (5 minutes each time). Converter-Peroxidase (POD) solution was added and incubated at 37°C for 30 minutes, and then washed in PBS for three times (5 minutes each time). Staining was performed using Diaminobenzidine (DAB) stain kit (Roche) at room temperature for 10 minutes, and was then washed in PBS for 5 minutes three times. The section was counterstained with hematoxylin for ten seconds and rinsed with water. The section was dehydrated with gradient alcohol, then dried and mounted with neutral gum. The section was observed under an optical microscope. Brown-stained nuclei represented positive staining. Blue-stained nuclei represented counterstaining by hematoxylin.A total protein extraction kit  was used to extract total protein. Procedures were followed according to the kit manual. The OD  values of the samples were measured using Coomassie Brilliant Blue G250. The concentrations of the protein of the samples were adjusted to be the same level (4-8 μg/μl). Bio-Rad  standard wet transfer apparatus and Poly vinylidene fluoride membrane (PVDF)  were used to transfer the proteins from the gel to the membrane. A constant flow (200 mA) was applied in the process of transfer and the transfer time was 60-120 minutes according to the molecular weight of the target proteins. The membrane was placed into TBST after transfer and rinsed for 1-2 minutes. Blocking buffer, which contained 5% skimmed milk powder, was added to block at room temperature for 60 minutes. The PVDF membrane was incubated in blocking buffer which contained corresponding primary antibody at 4°C overnight. The membrane was washed with TBST three times for 5 minutes each. Then the PVDF membrane was incubated in blocking buffer which contained the diluted secondary antibody at room temperature for 1 hour, then washed with TBST three times for 15 minutes each. A Beyo ECL Plus chemiluminescence kit  was used for staining in a darkroom. Finally, routine exposure and development were performed using X-ray film.ΔΔCT method was used for relative quantification.MiRanda and TargetScan were used to predict the target miRNAs of Uc.173. Default parameters of the software were applied and the results were not filtered. Only miRNAs predicted by both software were reserved and analyzed using the RegRNA platform. The expression of the miRNAs was detected by qRT-PCR. The 2-P < 0.05 represents a statistically significant difference and P < 0.01 represents a highly statistically significant difference.SPSS17.0  was used for data analysis. One-way ANOVA was used to compare the following three differences: the expression of Uc.173 in lead-exposed N2a cells, the gray values of the expression of the proteins, and the expression of miRNAs in lead-exposed nerve cells. A paired t-test was used to compare the difference of lead concentration in tissue or serum in the lead-induced nerve injury animal model. A Mann-Whitney test was used to compare the difference of serum lead concentration in different populations. A value of


VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT—were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plants, especially at full maturity stage. Metabolomic analysis by reverse phase LC-QTOF-MS confirmed several kaolin-induced modifications including a significant increase in the quantities of several secondary metabolites including flavonoids and anthocyanins in the latter ripening stages, probably resulting from the general stimulation of the phenylpropanoid and flavonoid pathways.Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways— Vitis vinifera L.) is a perennial woody plant with a great impact in the global economy, abundantly cultivated in areas with Mediterranean climates and spreading across temperate to semi-dry areas. Abiotic conditions, such as soil and atmospheric humidity, intense drought, and temperatures, have high impact on grape yield and wine quality 4, is an inert clay mineral that reflects potentially damaging ultraviolet and infrared radiation and transmits photosynthetically active radiation, resulting in leaf temperature decrease and photosynthetic efficiency increase  and stilbenoid pathways. Metabolomic analysis by reverse phase LC-QTOF-MS was also performed to unveil kaolin-induced modifications on several important secondary metabolites in the latter ripening stages.Vitis vinifera L.) grafted onto 110-R from the commercial vineyard “Quinta do Vallado,” in the Douro Demarcated Region (Denomination of Origin Douro/Porto), located at Peso da Régua, Portugal (41°09′44.5″N 07°45′58.2″W). The climate is typically Mediterranean-like, with a warm-temperate climate and dry and hot summers, with higher precipitation during winter but very low during the summer  and precipitation values (April to October) were reported in Dinis et al.  Kaolin , according to previous work done by our team , 3rd September (mature), and on 12th September (full mature); and immediately frozen in liquid nitrogen. In all ripening stages, sampling was performed in sunny and relatively hot days, so with relatively similar environmental conditions in all sampling dates. In the sampling procedure, 50–60 berries (about 5 per cluster) were collected always at the same time of the day, at 6.30 p.m. Phenological parameters of control vs. kaolin-treated fully-mature berries, respectively, were as follows: average berry weight—1.89 vs. 1.88 g, pH—3.98 vs. 3.94; total sugars—198.6 vs. 203.6 g/L. No apparent differences in skin to pulp ratio were noticed between control and kaolin-treated berries. The average water contents of control vs. kaolin-treated berries were as follows: 93.1 vs. 94.1% at green stage, 80.9 vs. 80.7% at veraison, 77.0 vs. 75.9% at mature stage, 74.5 vs. 74.4% at full maturation. For véraison sampling, a representative mix of colored and non-colored berries was obtained. This precaution procedure was adopted both in the cluster and for different clusters of the plant, with half of colored and half of non-colored berries collected from each condition and used for each experiment. The véraison rate was apparently similar between conditions with no apparent phonological displacement. No difference on véraison proportion between treatments was observable.Grape berry samples treated with kaolin and without treatment, i.e., control, were randomly collected from different positions in the clusters of different plants from different rows in the vine at four ripening stages: on 23th July (late green phase), 21st August  System  using reverse phase (RP) combined with positive ion ESI mode. A Zorbax Eclipse XDBC18 column  was used at 45°C and flow rate 0.6 mL/min with solvent A—water with 0.1% formic acid, and solvent B—acetonitrile with 0.1% formic acid. The gradient initiated from 25 to 95% B in 35 min, and returned to starting conditions in 1 min, with there-equilibration with 25% B for 9 min. For data acquisition, the TOF mass range was set from 50 to 1000 m/z. During the analysis two reference masses: 121.0509 m/z (C5H4N4) and 922.0098 m/z (C18H18O6N3P3F24) were continuously measured for constant mass correction and thus obtain the accurate mass. The capillary voltage was 3000 V with a scan rate of 1.0 scan per second. The nebulizer gas flow rate was 10.5 L/min.Metabolite data were normalized using the dry (lyophilized) weight (DW) of the samples. For all experimental conditions, three independent and randomized runs were performed in all metabolomic analysis.A total of 200 mg of grape berry tissue (without seeds) previously grounded in liquid nitrogen was used for total RNA extraction following the protocol by Reid et al.  in combiVvACT1 (actin), and VvGAPDH  were selected, as these genes were proven to be very stable and ideal for qPCR normalization purposes in grapevine , 5 mM dithiothreitol (DTT), and 0.1% (v/v) Triton X-100. The homogenates were thoroughly mixed and centrifuged at 18000xg for 20 min and the supernatants were maintained on ice and used for all enzymatic assays. Total protein concentrations of the extracts were determined by the method of Bradford  as substrate in 50 mM Tris-HCl pH 8.9. The rate of conversion of L-phenylalanine to cinnamic acid was monitored continuously in the spectrophotometer at 290 nm. Reactions were initiated by the addition of L-phenylalanine.PAL biochemical activity was determined in crude enzymatic extracts following the trans-cinnamic acid production at 41°C, in a total volume of 2 mL. The reaction mixture contained 0.2 mL of enzyme extract, 3.6 mM NaCl, and 25 mM L-phenylalanine .FLS biochemical activity determination was performed as described by Li et al.  with som−1 cm−1).The biochemical activity of UFGT was determined as described by Mori et al. , with sog for 20 min. Twenty μL of each supernatant were added to 1.58 mL of deionized water and 100 μL of Folin reagent, vigorously shaken and incubated for 5 min in the dark before adding 300 μL of 2M sodium carbonate. After 2 h of incubation in the dark, the absorbance of the samples was measured at 765 nm. Total phenolic concentrations were determined using a gallic acid calibration curve and represented as gallic acid equivalents (GAE). Anthocyanins were extracted from 150 mg of grape berry grounded tissue with 1 mL of 100% acetone. The suspension was vigorously shaken for 30 min. The homogenates were centrifuged for 20 min at 18000xg and the supernatants were collected. Anthocyanin extracts were diluted 1:10 in 25 mM potassium chloride solution pH 1.0 and absorbance was measured at 520 nm and 700 nm, using 25 mM potassium chloride solution pH 1.0 as blank. Total anthocyanin quantification was calculated in relation to cyanidin-3-glucoside equivalents, calculated by equation 1, per DW:MW is the molecular weight of cyanidin-3-glucoside (449.2 g mol−1), DF is the dilution factor and ε is the molar extinction coefficient of cyanidin-3-glucoside (26900 M−1 cm−1).The concentration of total phenolics and anthocyanins was performed as described in our previous work . For each condition, statistical differences between mean values are marked with asterisks .The results were statistically analyzed by Student's In a very recent study  in the phenylpropanoid pathway, than berries from the control vines , that encodes an enzyme catalyzing the first step in the phenylpropanoid pathway in which trans-cinnamic acid is produced, increased in the final maturation stages by 33% in berries from kaolin-treated plants. Here, we observed that VvPAL1 transcripts also appeared to be slightly more abundant in berries from kaolin treated plants at the late-green and véraison stages  that catalyzes the second reaction in the phenylpropanoid pathway, were also increased in berries from kaolin-treated plants, by 100% and approximately 20% at the mature and full mature stages, respectively.As the highest s Figure . As showstilbene synthase 1 (VvSTS1), that encodes the first enzyme of this pathway, was performed. However, with the primer pair used for amplification, we actually amplified several STS family genes, which can provide a broader sense of the changes in this metabolic pathway. Stilbene synthase (STS) is responsible for the condensation of 4-coumaroyl-CoA with 3 molecules of malonyl-CoA producing resveratrol. The real-time qPCR analysis revealed that VvSTS transcript levels increase up to 1000-fold from mature to full mature stages, but kaolin application appeared to stimulate VvSTS transcription only in the mature stage . However, kaolin application also stimulated VvCHS1 transcription at the mature and full mature stage in a very subtle way, as we had reported before . As shown in Figure VvFLS1 was mostly expressed at the véraison and mature stages  is the first enzyme of the flavonol biosynthetic branch of the flavonoid pathway. Gene expression analysis by qPCR revealed that s Figure , and thes Figure VvDFR1 expression was significantly higher in berries from vines treated with kaolin  catalyzes the final step of anthocyanin biosynthesis. The transcript levels of VvUFGT were noticeably higher in kaolin-treated than in control berries, particularly in the green (two-fold) and the mature (80%) stages, but also slightly, yet not statistically significant, in fully mature berries  is responsible for the first committed step in the pathway leading to the synthesis of flavan-3-ols (or flavanol) compounds, a group that comprises catechin, epicatechin, epigallocatechin, other tannins and proanthocyanidins; and also in the pathway that culminates with the synthesis of anthocyanins. We observed that, in all developmental stages, n Figure , with ins Figure . In agres Figure .VvMYB5b encodes a protein belonging to the R2R3-MYB family of transcription factors that has been unequivocally characterized as a regulator of the flavonoid pathway and as having a great role in anthocyanin- and proanthocyanidin-derived compounds accumulation  in mature berries from kaolin-treated vines, and approximately two-fold higher than the control in the green stage  do indeed provide strong evidence of a final increased biochemical activity. This increased biochemical activity of PAL, that is the result from the joint activity of all isoenzymes, may account for the observed lower levels of L-phenylalanine content in berries from kaolin-treated vines. Dai et al.  were successfully identified in the metabolomic analysis by LC-QTOF-MS and both were more abundant in berries from kaolin-treated vines especially at the latter ripening stages. This is in agreement with the enhanced flavonol biosynthetic pathway observed in berries from kaolin-treated vines, in particular at the mature stage. At that point, VvCHS1 expression in green berries from kaolin-treated plants, the stage in which its expression was the highest, also suggests that an enhancement of this metabolic step that begins the flavonoids pathway could have played a role in the higher phenolics concentration observed in this phase.The four-fold increase in VvUFGT, that glycosylates anthocyanidins/flavonols into anthocyanins using UDP-glucose as co-substrate, was indeed more expressed in mature berries from kaolin-treated vines, with a very good correlation with increased total UFGT higher enzymatic activity, just like the case of PAL and FLS, suggesting the increase in the transcription and activity of UFGT was key for increased anthocyanin concentrations. Like in the case of PAL, the enzymatic activity of UFGT is the clear-cut result from the joint action of all UFGT isoenzymes. Upstream, VvDFR expression was also enhanced in berries from kaolin-treated vines at the latter ripening stages, suggesting a whole stimulation of the anthocyanin synthesis pathway. Catechins/epicatechins, procyanidin B2, a proanthocyanidin, and the anthocyanin peonidin-3-galactoside were all also present in higher amounts in mature and fully mature berries from kaolin treated vines. This is in perfect agreement with the overall stimulation of phenylpropanoid and flavonoid pathways by foliar kaolin application. Moreover, anthocyanin stabilization and transport into the vacuole was also increased in berries during the major color change phases (véraison and mature) from kaolin-treated vines as demonstrated by increased VvGST4, VvMATE1, and VvABCC1 transcripts.Anthocyanins are responsible for berry color being, thus, an important quality trait of both fruit and red wine production. At the mature stage, berries are actively synthesizing anthocyanins in a process that stagnates in the very final ripening stage when the berries are ready for harvest. Fully mature berries from kaolin-treated vines had significantly more anthocyanins, in a process that appeared to be initiated in the mature phase. This difference could be explained by higher expression of genes involved in anthocyanin biosynthesis and accumulation in the latter ripening stages of berries from kaolin-treated vines. Anthocyanin accumulation in the grape berry is known to be impaired by high temperatures , and by Portuguese national funds (FCT-Portuguese Foundation for Science and Technology) under the project FCOMP-01-0124-FEDER-022692. AC was supported by Enoexcel—Norte—07-0124-FEDER-000032 and INTERACT - NORTE-01-0145-FEDER- 000017.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Brain abnormalities were assessed in 32 neonatal male rats (8 per group). Ex vivo T2-weighted imaging and diffusion tensor imaging (DTI) were performed using 11.7-T MRI. The expression of markers of myelin production , nonpyramidal cells , and pyramidal cells (staining of the nonphosphorylated heavy-chain neurofilament SMI-32) were histologically evaluated. Decreased brain volume, increased ventricle volume, and thinner cortices were observed by MRI in irradiated rats. However, no abnormalities in the cortical 6-layered structure were observed via KB staining in radiation-exposed rats. The DTI color-coded map revealed a dose-dependent reduction in the anisotropic signal , which did not represent reduced numbers of pyramidal cells; rather, it indicated a signal reduction relative to the vertical direction because of low nerve cell density in the entire cortex. We conclude that DTI and histological experiments are useful tools for assessing cortical and hippocampal abnormalities after prenatal exposure to radiation in rats.We assessed brain abnormalities in rats exposed prenatally to radiation (X-rays) using magnetic resonance imaging (MRI) and histological experiments. Pregnant rats were divided into 4 groups: the control group ( Many studies of survivors of prenatal exposure to the atomic bombings of Hiroshima and Nagasaki have shown that exposure to ionizing radiation during pregnancy has harmful effects on the development of the human central nervous system (CNS) Anisotropic water diffusion in neural fibers is more prominent in white matter (WM)-rich tissues Our goal was to use DTI to assess the brain abnormalities and changes in cellular organization induced by prenatal exposure to radiation, focusing on the cortex and hippocampus. In addition, a histological analysis was performed to evaluate the markers of myelin production , nonpyramidal cells , and pyramidal cells (SMI-32 staining) in this model.n = 3) and 3 radiation-exposed (n = 9) groups. The radiation-exposed groups underwent a single exposure to whole-body X-ray irradiation at a dose of 0.5 (n = 3), 1.0 (n = 3), or 1.5 Gy (n = 3), respectively, on day 15 of pregnancy Pregnant female Sprague–Dawley (SD) rats  were allowed to rest for 1 week before the experiment. The animals had free access to food and water and were kept under standard laboratory conditions of 22–23°C, ∼50% humidity, and a 12/12-h light/dark cycle. The 12 pregnant female rats were divided into 4 groups, comprising 1 control  4-week-old control rats , (2) 4-week-old rats exposed prenatally to 0.5 Gy of radiation , (3) 4-week-old rats exposed prenatally to 1.0 Gy , and (4) 4-week-old rats exposed prenatally to 1.5 Gy . Regardless of the prenatal conditions (control or radiation exposure), all neonatal rats were kept with their mothers in regular light/dark cycles until the MRI experiments were performed at 4 weeks after birth. MRI experiments were performed in all 8 rats per group, and histological experiments were performed in 4 rats per group.This study was carried out in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments at the University of Osaka (Permit Number: 23-040-0).2-weighted MR imaging  was conducted. Diffusion imaging was performed on all rats, using SE multi-shot echo-planar imaging . The DTI reconstruction and fractional anisotropy (FA) calculation were performed with the manufacturer-supplied software . The DTI fiber orientation was determined with reference to the rat brain atlas of Paxinos and Watson Before MRI scanning, all 4-week-old rats were euthanized via an overdose of pentobarbital  and prepared for MRI experiments by perfusion-fixation with saline containing heparin, followed by 7.5% paraformaldehyde, for 1 month. All brains were extracted. MRI experiments were performed on an 11.7-T MRI scanner  and a volume radiofrequency (RF) coil with an inner diameter of 25 mm was used for transmission and reception . The center of the imaging slices was carefully set at bregma −0.36 mm with reference to the rat brain atlas of Paxinos and Watson 2O2 in PBS containing 0.1% Triton X-100 to inactivate endogenous peroxidase, and were then incubated with the Antigen Retrieval Reagent UNIVERSAL  for 30 min at 90°C. After washing with PBS, sections were incubated with a rabbit anti-calbindin D-28k (CaBP) polyclonal antibody , or a mouse anti-SMI-32 monoclonal antibody , containing 10% normal goat serum at 4°C overnight. After incubation, the sections were rinsed with PBS and incubated with biotinylated anti-rabbit IgG or biotinylated anti-rat IgG. The immunoreactive products were visualized with a Vectastain ABC elite kit  using 0.01% 3,3′-diaminobenzidine tetrachloride (Sigma) in 0.03% H2O2 as a chromogen. KB staining for myelin was carried out The brains were removed from the skull, and were embedded in Optimal Cutting Temperature (OCT) compound, with reference to the rat brain atlas of Paxinos and Watson P<0.05 was considered significant.All statistical analyses were performed using Prism 5 . One-way analysis of variance (ANOVA) with Dunnett was used to compare changes against control animals in the FA value, brain volume, ventricle volume, and cortical thickness across groups of radiation-exposed animals. Correlation analysis between radiation doses and MRI were performed using Pearson product-moment correlation analysis. 2WI results for all groups are shown in rd ventricle volume of rats at the same age exposed prenatally to the same three levels of radiation dose was larger than that of the control rats. The cortices were also thinner than those of the control rats. The CC and EC of rats exposed to 0.5 and 1.0 Gy of radiation prenatally were thinner than those of the control rats. These results show decreased brain volume, increased ventricle volume, thinner cortex, thinner CC, and thinner EC in irradiated rats and all effects were dose dependent.Typical THistological sections around the S1 cortex stained with KB, CaBP, and SMI-32 are shown at similar magnifications in P<0.0001, P<0.0001, P<0.0001, P = 0.004, P<0.0001, P<0.0001, P = 0.005, P<0.0001, 2WI, DTI, and histological staining for myelin (KB), nonpyramidal cells (CaBP), and pyramidal cell (SMI-32).This report evaluated the presence of cortical and hippocampal abnormalities in rats prenatally exposed to radiation, using TThe effects of irradiation on the developing rodent CNS have been discussed for many years. Many macroscopic and microscopic analyses have reported that rodent brains exposed prenatally developed microcephaly, ectopic gray matter, reduction of cortical thickness, abnormal neuronal migration, and disturbance of synaptogenesis Miki et al. reported that the brain weight of rats X-ray irradiated prenatally was significantly lower than that of control rats. Similarly, at 4 weeks after birth, the brain volume of rats exposed to X-ray irradiation prenatally was dose-dependently smaller than that of control rats, as shown by MRI. This observation of brain volume as evaluated using MRI is in agreement with the results of a previous histological study A previous work noninvasively characterized the regional postnatal radiation-induced developmental alterations observed in the mouse brain, using longitudinal MRI The comparison of the DTI color maps of the control group with those of the radiation-exposed groups revealed an obvious decrease in green fiber densities in the radiation-exposed rat cortex. The thickness of layers I–III (SL) in radiation-exposed rats was dose-dependently smaller than that of control rats. However, a 6-layered structure was observed in all control and irradiated rats (0.5–1.5 Gy), as assessed by KB staining. Similarly, CaBP-positive cells were observed in the neuropil of SL and in pyramidal cells throughout the cortex in all rats. In addition, SMI-32-positive cells were observed at the cell body and dendrite areas of pyramidal cells, which allowed a clear distinction between layer III (OL) and layer V (IL) in control and irradiated rats. However, IL also became dose-dependently thinner, so that layer-specific abnormalities were not seen in the cerebral layered structure of radiation-exposed rats. The FA values in the cortex of 4-week-old rats exposed to 1.0 or 1.5 Gy of radiation were dose-dependently smaller than those of the control rats. These observations were in agreement with the findings from KB staining. Moreover, a dose-dependent reduction in the anisotropic signal (green) in the vertical direction was observed in DTI, which was not mainly due to abnormal axonal fiber extension. A 6-layered structure was observed in all control and irradiated rats (0.5–1.5 Gy) using KB, CaBP, and SMI-32 staining. These observations did not represent an absence of pyramidal cells; rather, they reflected the reduction in the signal relative to the vertical direction because of a decrease in a real nerve cell density in the entire cortex. In addition, the FA values of rats that were exposed to X-ray irradiation prenatally were dose-dependently smaller than those of control rats, as shown by DTI in the CC and EC. Abnormalities of CC and EC WM structures were observed in all irradiated rats (0.5–1.5 Gy), as assessed using KB staining. We suggest that these abnormalities in the WM of irradiated rat brains detected by KB staining are consistent with the reduction of FA values assessed by DTI.Prominent hippocampal atrophy was observed in the brains of rats that were exposed to radiation prenatally. Sun et al. also detected neocortical ectopias after prenatal X-irradiation, as assessed using histological examination This study found that decreased brain volume, increased ventricle volume, and reduced cortical and white matter thickness in irradiated rats can be assessed using MRI. Rats that were irradiated at embryonic day 15 day exhibited postnatal changes such as thinner cerebral cortex, decreased size of the whole brain, and increased ventricle volume. Comparison of the DTI color maps obtained here revealed decreased horizontal color fiber densities in the S1 cortex of radiation-exposed rats. However, no abnormalities in the cortical 6-layered structure were observed in any of the radiation-exposed rats. Moreover, ectopic neurons were observed only in the hippocampus  of rats exposed to 1.5 Gy of radiation prenatally. DTI is a useful tool for examining and quantifying cortical and hippocampal abnormalities in model rats exposed to radiation prenatally.


Accumulating evidence has shown that up-regulation of microRNA-25(miR-25) is associated with the prognosis of several types of human malignant solid tumors. However, whether miR-25 expression has influence on the prognosis of hepatocellular carcinoma (HCC) is still unknown.The differentially expressed amount of the miR-25 was validated in triplicate by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Survival rate was analyzed by log-rank test, and survival curves were plotted according to Kaplan–Meier. Multivariate analysis of the prognostic factors was performed with Cox regression model.The expression of miR-25 was significantly upregulated in HCC tissues when compared with adjacent normal tissues (p<0.0001). Patients who had high miR-25 expression had a shorter overall survival than patients who had low miR-25 expression . The multivariate Cox regression analysis indicated that miR-25 expression , TNM stage , and vein invasion  were independent prognostic factors for overall survival.Our data suggests that the overexpression of miR-25 in HCC tissues is of predictive value on poor prognosis.http://www.diagnosticpathology.diagnomx.eu/vs/1989618421114309The virtual slide(s) for this article can be found here:  Hepatocellular carcinoma (HCC) is one of the most malignant solid tumors and is the second leading cause of cancer-related mortality . The estThe microRNAs (miRNAs) are a class of highly conserved short noncoding RNAs, which suppress protein expression by inhibiting translation or inducing mRNA degradation by binding to the 3′UTR of target mRNAs,11. BeyoMiR-25 is a member of the miR-106b~25 cluster, which includes miR-106b, miR-93 and miR-25, that is located within intron 13 of the minichromosome maintenance protein 7(MCM7) gene on chromosome 7q22.1 ,15. AccuRecently, Li Y et al. found that the miR-106b~25 cluster was overexpressed in HCC tissues as well as cell lines, suggesting an important role of miR-106b~25 cluster in carcinogenesis and development of HCC . HoweverA total of 131 fresh specimens of HCC and paracarcinomatous liver tissues were obtained from 131 patients who underwent hepatic resection at Provincial Hospital affiliated to ShanDong University between June 2006 and July 2012. Among all the subjects, the mean follow-up period was 47 months (from 11 months to 65 months), and there was no censored case. All these tissues were immediately stored in liquid nitrogen and kept freshly frozen at − 80°C. Written informed consent was accomplished from the patients for publication of this study and any accompanying images. Study protocol was approved by the Ethics Committee of Provincial Hospital affiliated to ShanDong University. Before surgical therapy, none of the patients had received neoadjuvant chemotherapy, radiation therapy, or immunotherapy. Related important clinical information, including age, gender, serum level of α-fetoprotein (AFP), liver cirrhosis, Hepatitis B virus infection, tumor diameter, number of tumor nodules, and TNM stage were collected from each patient’s medical records. Clinical staging was performed according to the 2002 American Joint Committee on Cancer/International Union Against Cancer TNM staging system [T) value was calculated. The 2-ΔCT (ΔCT=CTmiR25-CTU6 RNA) method was used to quantify relative amount of miR-25.Total RNA was isolated from frozen specimen by homogenizing tissue in Trizol reagent , according to the manufacturer’s instructions. The purity and concentration of RNA were determined using NanoDrop 1000 spectrophotometer . The differentially expressed amount of the miR-25 was validated in triplicate by quantitative reverse-transcription polymerase chain reaction (qRT-PCR). Briefly, 2 ug of RNA was added to RT reaction, and then, the cDNA served as the template for amplification of PCR with sequence-specific primers  using SYBR PrimeScript miRNA RT-PCR kit  on the 7500 Real-Time PCR systems . The PCR cycling profile was denatured at 95°C for 30 s, followed by 40 cycles of annealing at 95°C for 5 s, and extension at 60°C for 34 s. Small nucleolar RNA U6 was used as an internal standard for normalization. The cycle threshold  and GraphPad Prism 5 . The comparison of the expression level of miR-25 between HCC tissues and adjacent normal tissues was performed using the two-sample Student’s t test. The correlation between the expression level of miR-25 and clinicopathological characters was assessed with the two-sample Student’s t test. Survival rate was analyzed by log-rank test, and survival curves were plotted according to Kaplan–Meier. Multivariate analysis of the prognostic factors was performed with Cox regression model. All tests were two tailed and results with P<0.05 were considered statistically significant.The expression of miR-25 was significantly upregulated in HCC tissues  when compared with adjacent normal tissues, age (p=0.21), liver cirrhosis (p=0.17), Hepatitis B virus infection (p=0.09), vein invasion (p=0.24), tumor diameter (p=0.13), or number of tumor nodules (p=0.51).Considering that the level of miR-25 expression was significantly correlated with TNM stage and serum AFP level, we hypothesized that miR-25 might affect the prognosis of HCC patients. To confirm this possibility, the miR-25 expression level and the prognosis of patients with HCC were analyzed by using the Kaplan–Meier method. The results indicated that patients who had high miR-25 expression had a shorter overall survival than patients who had low miR-25 expression (median overall survival, 31 months versus 42.9 months, p=0.0192; Figure Although a growing number of novel treatment strategies have been developed for HCC, such as molecular targeted therapy and gene therapy, to our disappointment, satisfactory therapeutic outcomes have not been achieved ,22. ConsThe discovery that noncoding components of the genome, including microRNA, can contribute to the pathogenesis of cancer has led investigators to contemplate using these molecules to guide clinical decision making . So far,Li Y et al. have found that miR-25 was strongly up-regulated in HCC tissue when compared with the corresponding paired non-tumor samples. However, the clinical significance of miR-25 gene expression in HCC remains unclear. In the present study, we found that miR-25 expression was proven to be associated with advanced TNM stage, suggesting that miR-25 might be involved in the carcinogenesis and metastasis of HCC. More importantly, we proved that patients with a high expression of miR-25 tended to have shorter survival than patients with lower levels, indicating that high miR-25 level is a marker of poor prognosis for patients with HCC. However, the precise molecular mechanisms behind the altered expression of miR-25 in HCC and its function are not very clear. In the study by Li Y et al., knock-down studies for the miR-106b-25cluster, which includes miR-106b, miR-93 and miR-25, showed that the expression of the cluster was necessary for cell proliferation and for anchorage independent growth . AdditioIn summary, to the best of our knowledge, the present study is the first to report the differential expression of miR-25 in HCC and the possible use of miR-25 as a novel prognostic marker in HCC. The present findings demonstrate high expression of miR-25 in HCC tissue, which is associated with a poor prognosis in HCC patients. Further studies are needed to elucidate the mechanisms of action of miR-25 in HCC.The authors declare that they have no competing interests.ZXS and JZ carried out the experimental studies and drafted the manuscript. ZHR, WMG, and YGW carried out part of the experimental studies. CKQ designed the experiments and modified the manuscript. All authors read and approved the final manuscript.


BCR-ABL1 transcripts are crucial for managing patients with chronic myeloid leukemia (CML). Although real-time quantitative polymerase chain reaction (RT-qPCR) can be measured on an International Scale (IS), this has not become fully universal. By using a WHO international standard panel established for calibrating secondary standards based on the IS, we have previously developed an RT-qPCR kit, ODK-1201, for quantification of major BCR-ABL1.Detection and quantitation of BCR-ABL1IS.In this study, the reliability of kit-specific conversion factor 1.12 was validated by exchanging patients’ samples between three local clinical laboratories and a reference laboratory. The mean bias of the local method after IS conversion was 1.6 fold lower than the reference method. The clinically-useful sensitivity of the kit was further evaluated for monitoring patients with deep molecular response. Based on the correlation of the IS values between ODK-1201 and the reference laboratory method, the detection level of the kit was estimated as 0.0032 % BCR-ABL1 on the IS in 2 h after RNA extraction, thus contributing to standardization of molecular monitoring in CML.ODK-1201 is a highly sensitive one-step RT-qPCR system for detecting  BCR-ABL is a constitutively active tyrosine kinase generated by the Philadelphia chromosome translocation, and is recognized as the underlying pathogenetic mechanisms of chronic myeloid leukemia (CML)  , as established in the IRIS trial  were carried out to identify of the performance of ODK-1201 and to get the laboratory-specific CF, sponsored by Otsuka Pharmaceutical Co. Ltd. Total 468 CML patients were recruited for these studies, and peripheral blood samples were taken after written informed consent in 24 institutions in Japan, between July 2012 and October 2013. These studies were conducted in accordance with the Declaration of Helsinki, and its design was approved by each hospital’s Institutional Review Board. The samples were firstly analyzed in three commercial laboratories, BML, Inc., SRL, and LSI Medience Corporation  in Japan, and thereafter in SA Pathology, Adelaide, Australia, a recognized reference laboratory  was added to a mixture of 13 μL of mix solution (R1) for RT-qPCR and 2 μL of Mn(OCOCH3)2 solution and applied to ABI™ 7500 Fast Dx system. The assay conditions have been described previously , and analyzed according to the suggested method for calibration of secondary standards (http://www.nibsc.org/documents/ifu/09-138.pdf). The panel comprises four ampoules, each containing freeze-dried BCR-ABL1-positive and -negative cells at various ratios from 0.01 to 10 % BCR-ABL1IS. RNAs were isolated from each ampoule with a QIAamp RNA Blood Mini Kit (QIAGEN) and were subjected to the measurement of major BCR-ABL1 and ABL1 mRNAs with the ODK-1201 kit. The CF of the ODK-1201 was calculated by comparing the values of % BCR-ABL1/ABL1 obtained with the kit with those from the WHO standard panel. The RT-qPCR method carried out at the Adelaide reference laboratory has been previously detailed  in BML, RNeasy Mini Kit (QIAGEN) in SRL, or RNeasy Lipid Tissue Mini Kit  in LSI, respectively. The study diagnostic kit in this study was coded ODK-1201. It is based on a HawkZ05 Fast One-Step RT-qPCR Kit . In this kit, reverse transcription and quantitative PCR are performed in one tube. The analysis was carried out on an ABI™ 7500 Fast Dx system . In the quantitation with this kit, specific regions of major The ODK-1201 kit-specific CF was 1.12 determined by using the WHO standard panel by following the method for calibration of secondary standards as outlined in White et al. . The calBCR-ABL1 values from the reference and the local laboratories were converted to the IS by multiplying by their specific CFs. The samples with positive BCR-ABL1 values were converted to the IS using the CF 1.12 and compared , and then sent to the reference laboratory. None of the samples was degraded. The BCR-ABL1 value of 0.10 % BCR-ABL1IS) between the reference method and the ODK-1201was determined as 84 % (N = 43/51) of samples.The overall concordance of the data between ODK-1201 and the reference laboratory systems was acceptable Fig. . After cBCR-ABL1IS. To verify the clinically-useful sensitivity of the ODK-1201 kit, we tested the correlation between the two methods in the assessment of 46 clinical samples with IS between the lowest positive value (LPV) and 0.1 % BCR-ABL1IS. A high correlation coefficient (r = 0.89) was found  to standardized baseline (as 100 % BCR-ABL1IS) established in the IRIS study, but not deeper molecular response . In contrast, when BCR-ABL1-negative samples were analyzed by the kit, BCR-ABL1 transcripts was never detected, suggesting low false positivity of the kit (data not shown). Of note, the measurement can be carried out using only 7 mL of blood, a sample which is smaller than the previously recommended volume (Baccarani et al. The achievement of MMR has been recognized as the therapeutic goal for chronic-phase CML patients based on the results reported on the IRIS trial and subsequent ones (Baccarani et al. BCR-ABL1 using commercial kits.The variation of results obtained by RT-qPCR is due to each stage of the procedure, including sample collection, transportation time from clinics to laboratories, RNA extraction, reverse transcription, and the actual quantitative PCR (Branford et al. BCR-ABL1 based on the IS, which contributes a useful tool for evaluation of deep molecular responses and standardization of treatment in CML.ODK-1201 is a rapid one-step high sensitivity RT-qPCR system for detecting


Kinetic parameters revealed that only derivatives labeled closer to the N-terminus  were hydrolyzed by ACE, indicating that peptides bearing the TOAC moiety far from the ACE cleavage site (Phe8-His9 peptide bond) were susceptible to hydrolysis, albeit less effectively than the parent compound. CD spectra indicated that AngI exhibited a flexible structure resulting from equilibrium between different conformers. While the conformation of N-terminally-labeled derivatives was similar to that of the native peptide, a greater propensity to acquire folded structures was observed for internally-labeled, as well as C-terminally labeled, analogues. These structures were stabilized in secondary structure-inducing agent, TFE. Different analogues gave rise to different β-turns. EPR spectra in aqueous solution also distinguished between N-terminally, internally-, and C-terminally labeled peptides, yielding narrower lines, indicative of greater mobility for the former. Interestingly, the spectra of peptides labeled at, or close, to the C-terminus, showed that the motion in this part of the peptides was intermediate between that of N-terminally and internally-labeled peptides, in agreement with the suggestion of turn formation provided by the CD spectra. Quenching of the Tyr4 fluorescence by the differently positioned TOAC residues corroborated the data obtained by the other spectroscopic techniques. Lastly, we demonstrated the feasibility of monitoring the progress of ACE-catalyzed hydrolysis of TOAC-labeled peptides by following time-dependent changes in their EPR spectra.Conformational properties of the angiotensin II precursor, angiotensin I (AngI) and analogues containing the paramagnetic amino acid TOAC  at positions 0, 1, 3, 5, 8, 9, and 10, were examined by EPR, CD, and fluorescence. The conformational data were correlated to their activity in muscle contraction experiments and to their properties as substrates of the angiotensin I-converting enzyme (ACE). Biological activity studies indicated that TOAC The renin–angiotensin system (RAS) exerts an important role in cardiovascular and hydro-electrolyte homeostasis –4. SeverCleavage of the C-terminal dipeptide is catalyzed by the metallopeptidase angiotensin I-converting enzyme EC 3.4.15.1, ACE) [.1, ACE [1H NMR studies in DMSO or H2O/TFE 34%/66% v/v evinced the existence of a turn in the C-terminal portion of AngI, which is stabilized in the former solvent [Interestingly, while an extensive literature has been produced concerning AngII and its active (agonists or antagonists) or inactive analogues, a much smaller amount of data exists focusing on AngI –15. More solvent –13. MoleStudies of the conformational properties of peptides have made use, among other spectroscopic techniques, of electron paramagnetic resonance (EPR) spectroscopy by means of incorporation of the non-coded paramagnetic amino acid TOAC  whose introduction allowed linking a spin label to the peptide backbone via a peptide bond . Initialα,α-tetrasubstituted cyclic structure hampers rotation about side chain bonds, leading to formation of bends in the peptide backbone [10-helical conformations [As a result, a large amount of applications have been reported in the literature , expandibackbone . Moreovermations –21.Structure-function studies of TOAC-carrying peptides have been extensively performed by our group, AngII being the first biologically active peptide investigated –18. Conf1R and BKRB1, containing binding sites for the hormones [8-His9 of AngI, releasing the vasoconstrictor AngII [A novel approach was undertaken by examining the interaction between the parent hormones AngII and BK, as well as their active and inactive TOAC analogues, and constructs of their respective GPCRs, AThormones –30. In ahormones . The enzor AngII . AlthougIn the present work we describe the synthesis of additional AngI analogues carrying TOAC at positions 0, 5, or 10. To our knowledge this is the first time a peptide is labeled at the C-terminus. Thus, an almost complete TOAC-scan was performed, where a total of seven TOAC-carrying analogs were evaluated from the point of view of their conformational properties using EPR, CD, and fluorescence spectroscopy. In order to look for structure-activity relationships the pharmacological activity of the peptides was examined in muscle contraction experiments (guinea pig ileum and rat uterus), according to ref. , and theTert-butyloxycarbonyl (Boc) and 9-fluorenylmethyloxycarbonyl (Fmoc) amino acids were purchased from Bachem  and purified rabbit lung ACE was from Sigma . Dimethylformamide (DMF) and ninhydrin were distilled over P2O5 and under reduced pressure, respectively. All solvents were HPLC grade and all chemicals met ACS standards. The molar concentration of rabbit lung ACE was determined by active site titration with lisinopril, as previously described [escribed –33.10-AngI peptide was synthesized starting from TOAC-copoly(styrene-1% divinylbenzene) support previously obtained by attaching Fmoc-TOAC to a chloromethyl-resin according to a standard protocol [TOAC-containing AngI derivatives were synthesized manually according to the combined Boc/Fmoc strategies as previously reported , 31. Allprotocol . The cru18-column) using aqueous 0.02 M ammonium acetate (pH 5) and 60% acetonitrile solutions as solvents A and B, respectively . Peptide homogeneity was determined through analytical HPLC, mass spectrometry and amino acid analysis. All peptides were obtained in satisfactory yield; the synthesis scale was 0.2 mmol.Unlabeled peptides were synthesized using the Boc strategy. The peptides were purified by preparative HPLC .RP-HPLC analyses were carried out in a TFA/acetonitrile gradient using a Waters Associates HPLC system consisting of two 510 HPLC pumps, an automated gradient controller, a Rheodyne manual injector, a 486 UV detector, and a 746 data module. The column employed was a Vydac C18 column , detection at λ = 210 nm, using the solvent systems A: 0.1% TFA/H18 . The elution was carried out with solvents A (0.1% TFA/H2O) and B (60% acetonitrile/0.1% TFA/H2O) at a flow rate of 0.4 mL/min using a linear gradient from 5% to 95% B in 30 min. The condition used for mass spectrometry measurements was a positive ESI. Specifically for the case of monitoring enzymatic reactions, a different linear gradient of 35 to 50% (v/v) of solution B in 15 min, with flow rate of 1.5mL/min was used.The liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) experiments were performed on a system consisting of a Waters Alliance model 2690 separations module and model 996 photodiode array detector  controlled with a Compaq AP200 workstation coupled to a Micromass model ZMD mass detector . The samples were automatically injected on a Waters narrow bore Nova-Pak column CPeptide composition was monitored using amino acid analysis performed on a Biochrom 20 Plus amino acid analyzer  equipped with an analytical cation-exchange column.B and τC) of the paramagnetic compounds were calculated from spectral line heights and line widths according to Schreier et al. [Spectra were obtained at 9.5 GHz in a Bruker ER 200 spectrometer at room temperature (22 ± 2°C) using flat quartz cells from Wilmad Glass Co., Buena, NJ, USA. The magnetic field was modulated with amplitudes less than one-fifth of the line widths, and the microwave power was 5 mW to avoid saturation effects. Rotational correlation times . Equivalent results were obtained in triplicate measurements.4 residue was excited at 275 nm. Equivalent results were obtained in triplicate measurements.Static fluorescence spectra were obtained at room temperature (22 ± 2°C) in a Hitachi F 2500 spectrofluorimeter , using cuvettes with excitation path lengths of 2 mm or 5 mm and emission path lengths of 10 mm. Excitation and emission slits were 5 nm and the peptides concentration was 100 μM in 0.02 M phosphate buffer, pH 7.0. The peptides Tyr2-5% CO2 and the temperature was kept at 30°C in order to inhibit the spontaneous contractions observed at higher temperatures.The biological potencies of AngI and their TOAC attaching derivatives were examined in rat uterus and guinea-pig ileum, accordingly to previous report . Briefly2−5% CO2 and the temperature was kept at 37 ± 0.5°C.For contractile studies in guinea-pig ileum, the lower portion of ileum was excised from guinea-pigs (220–250 g) and washed in Tyrode solution. Segments of 4-cm length were suspended in 5 mL organ baths which contained aerated Tyrode solution bubbled with a gas mixture of 95% OThe concentration-response curves were obtained by administration of successive treatments with the agonist for 90 s, at 5-min intervals. Intrinsic activities of paramagnetic analogues were estimated by the maximum response relative to that of parent peptide (AngI). As early determined , the bio2 at pH 7.0 . The hydrolysis reaction and all released peptide products were monitored by LC/ESI-MS as a function of time as already described [−5 M). The hydrolysis reaction was interrupted at different times by adding 0.1% TFA aqueous solution. The kcat, Km and kcat/Km values were obtained by analysis of the non-linear regression data using the GraFit program (Erithacus Software). The standard deviation of these parameters was less than 5%.The hydrolysis of peptides by purified rabbit lung ACE was performed at 37°C in 0.1 M Tris-HCl buffer containing 50 mM NaCl and 10 μM ZnClescribed , and theWe have previously reported studies of ACE’s enzymatic activity upon AngI and its analogues containing TOAC at positions 1, 3, 8, and 9 in conjunction with preliminary EPR and fluorescence data . Here we1-AngI [0-AngI analogue also retained partial biological potency, with two-fold and 18% that of AngI in rat uterus and guinea pig ileum, respectively. None of the other TOAC-AngI derivatives  showed biological potency, in close accordance with previous observations that the N-terminal portion of released AngII is less important for maintaining its potency [et al. who found that, with regard to biological activity, introduction of TOAC in the N-terminal part of neuropeptide Y, a GPCR ligand, was better tolerated than in proximity to the C-terminus [The peptides behavior in contraction experiments was determined in guinea pig ileum and rat uterus following standard procedures –25, 31.  potency –38. Thesterminus .8-AngI and TOAC9-AngI lack the ability to act as substrates, possibly due to the fact the TOAC residue occupies either position P1 or position P’1, respectively, of the AngI P1-P’1 scissile bond, C-terminally labeled TOAC10-AngI does not act as a substrate due to proximity of the spin label to the cleavage site, possibly creating steric hindrance. In this case, both the bulkiness of TOAC and the turn promoted by this residue (see below) would prevent fitting of the sequence to be cleaved to the enzyme’s active site.ACE’s peptidase activity upon AngI and its TOAC-containing derivatives was examined as described in Methods. The reaction progress was monitored through LC/EMI-MS of the components in solution. The results indicated that AngI derivatives carrying TOAC at positions 0, 1, 3, and 5 were cleaved by ACE, while the remaining analogues were not. While TOACcat/Km value for AngI was almost eight times higher than that for the weakest TOAC5-AngI substrate (15.4 μM-1.min-1vs 2 μM-1.min-1). These results are in accord with previous work [The kinetic parameters calculated for AngI and its TOAC derivatives  suggest ous work –41 that 8-, TOAC9-, and TOAC10-AngI analogues, when these peptides are exposed to ACE, they fail in being converted to the corresponding spin-labeled AngII. However, this is not the case for TOAC3- and TOAC5-AngI, since these analogues do act as substrates for the enzyme. These results indicate that although the latter analogues are converted to their AngII counterpart, these products are not accepted as ligands for AngII receptors. Thus, the active site of ACE is less selective than the receptor site where AngII binds to trigger muscle contraction.When muscle contraction experiments are examined in the light of ACE activity, it is seen that in the case of TOAC8-, TOAC9-, and TOAC10-AngI analogues, when these peptides are exposed to ACE, they fail in being converted to the corresponding spin-labeled AngII. However, this is not the case for TOAC3- and TOAC5-AngI, since these analogues do act as substrates for the enzyme. These results indicate that although the latter analogues are converted to their AngII counterpart, these products are not accepted as ligands for AngII receptors. Thus, the active site of ACE is less selective than the receptor site where AngII binds to trigger muscle contraction.When muscle contraction experiments are examined in the light of ACE activity, it is seen that in the case of TOAC9-AngI and TOAC10-AngI, but not in the spectra of N-terminally labeled TOAC0-AngI and TOAC1-AngI.C)  measured from the spectra of TOAC-labeled analogs. A slight difference is seen between the values for TOAC at the peptides N-terminus and in the other analogues. Although very small, this difference is in line with the more restricted motion in the backbone and in the C-terminal analogues and could be related to the greater flexibility of the N-terminal region, that, on the average, would expose more this region to the bulk aqueous phase, therefore, to a slightly higher polarity.Calculated rotational correlational times . On the other hand, TOAC8-AngI gave rise to a spectrum characteristic of an unordered structure in aqueous solution  were able to exert partial activity, in agreement with the known lesser importance of the N-terminal region for this activity [1-AngI-promoted muscle contraction is in agreement with the work of Cordopatis and Theodoropoulos who showed that Aib1-AngII displays high potency in thoracic rabbit aorta strips [As mentioned above, muscle contraction experiments indicated that only AngI analogues labeled at the N-terminus , and the EPR spectra of their TOAC counterparts are very similar and essentially indistinguishable.0/h-1 (ratio of heights of the mid-field and high-field lines) as a function of time. This parameter bears a correlation with the rotational correlation time, τC; the higher the correlation time, the higher h0/h-1 [C, is higher than that of the product  retained partial muscle contractile activity in guinea pig ileum and rat uterus. The lack of activity of derivatives carrying TOAC as residues 3–10 was likely due to the known turn-promoting properties of this cyclic disubstituted glycine. In contrast, the ability to serve as substrates in the ACE-catalyzed hydrolysis was observed for analogues containing TOAC up to residue 5. These findings indicated that the structural requirements for enzymatic hydrolysis are less selective than those regarding ligand-receptor binding and signal transduction.In this context, only analogues attaching TOAC at the N-terminal end . Moreover, centrally located residues yielded the highest values of rotational correlation times, pointing to the role of the backbone in restricting the motion of these residues.0-AngI and TOAC1-AngI) presented CD spectra similar to those of native AngI, suggesting a direct relationship between structure and function for these sequences. All other analogues, labeled either at internal positions or at the C-terminal region gave rise to spectra corresponding to more folded structures. The spectra were characteristic of different types of β-turns, pointing to the sequence-dependence of the type of fold originated by TOAC. A rather interesting event stems from the fact that even TOAC10-AngI gave rise to a CD spectrum suggestive of a β-turn, in agreement with 1H NMR data for AngI.Representative CD spectra of more flexible conformers were observed mainly for N-terminally labeled analogues. It is noteworthy that the biologically active analogues (TOAC4 fluorescence was observed for all peptides, except TOAC9-AngI. The degree of quenching paralleled the results found by EPR and CD, namely, besides the more effective quenching by the next neighbors, the more flexible N-terminal region was capable of greater quenching than the more rigid C-terminal region.The intramolecular quenching effect of paramagnetic TOAC upon TyrFinally, we used the time dependence of the EPR spectra to monitor the kinetics of ACE-catalyzed hydrolysis, in spite of the small difference between the spectra of the TOAC-labeled AngI analogues and those of their two-residue shorter products. All analogues containing the spin label inserted up to residue 5 were acceptable as ACE substrates. This approach should be useful in studies of reactions involving TOAC-labeled peptides.


This study was designed to validate the Dutch Physical Activity Questionnaires for Children (PAQ-C) and Adolescents (PAQ-A).2 peak).After adjustment of the original Canadian PAQ-C and PAQ-A , content validity of both PAQs was assessed and calculated using item-level (I-CVI) and scale-level (S-CVI) content validity indexes. Inter-item and inter-rater reliability of 196 PAQ-C and 95 PAQ-A filled in by both children or adolescents and their parent, were evaluated. Inter-item reliability was calculated by Cronbach’s alpha (α) and inter-rater reliability was examined by percent observed agreement and weighted kappa (κ). Concurrent validity of PAQ-A was examined in a subsample of 28 obese and 16 normal-weight children by comparing it with concurrently measured physical activity using a maximal cardiopulmonary exercise test for the assessment of peak oxygen uptake .For both PAQs, I-CVI ranged 0.67-1.00. S-CVI was 0.89 for PAQ-C and 0.90 for PAQ-A. A total of 192 PAQ-C and 94 PAQ-A were fully completed by both child and parent. Cronbach’s α was 0.777 for PAQ-C and 0.758 for PAQ-A. Percent agreement ranged 59.9-74.0% for PAQ-C and 51.1-77.7% for PAQ-A, and weighted κ ranged 0.48-0.69 for PAQ-C and 0.51-0.68 for PAQ-A. The correlation between total PAQ-A score and VO2 peak. Both PAQs have an acceptable to good reliability and validity, however, further validity testing is recommended to provide a more complete assessment of both PAQs.Both PAQs have an excellent content validity, an acceptable inter-item reliability and a moderate to good strength of inter-rater agreement. In addition, total PAQ-A score showed a moderate positive correlation with VOThe online version of this article (doi:10.1186/2049-3258-72-47) contains supplementary material, which is available to authorized users. Physical inactivity in children and adolescents has become a major issue in public health . In BelgVarious objective and subjective methods have already been suggested to evaluate the level of physical activity in children and adolescents . UnfortuThe self-report Canadian PAQ-C and PAQ-A, developed by Kowalski et al.  are valiIn Flanders (the Dutch speaking part of Belgium), however, the reliability and validity of these questionnaires has not yet been evaluated in children and adolescents. Therefore, the principal aim of this study is to assess the reliability and validity of a Dutch version of the PAQ-C and PAQ-A in children and adolescents.The study was conducted in accordance with the ethical rules of the Helsinki Declaration. Informed consent was obtained from all children and their parents or legal guardian. The study protocol was approved by the Antwerp University Hospital Ethics Committee .Both PAQs were evaluated by an expert committee consisting of three professionals in Pediatrics, one professional in Sports Medicine, and two professionals in Epidemiology and Sociology . The selection was based on the response rate of schools which were located in the vicinity of the university and/or home of the researchers involved. Children or adolescents and their parents were informed about this study by means of distribution of an information letter. Afterwards, all responders received a letter with detailed information on the study protocol and the appropriate physical activity questionnaires to be completed and returned. Parent–child reliability (i.e. inter-rater agreement) was evaluated based on the PAQs filled in by both child and parent, independently of each other.2 peak. During the CPET to volitional fatigue an electronically braked cycle  was used  [2) was collected breath-by-breath and averaged every 10 seconds. All subjects exercised to volitional exhaustion and achieved a peak respiratory gas exchange ratio (RER) >1.0.Adolescents who completed the PAQ-A on the same day as a cardiopulmonary exercise test (CPET) were included to study concurrent validity. These study participants were a subset of obese and normal-weight children recruited during 2012–2013 participating in a prospective study (METAFIT study) conducted at the Jessa Hospital Hasselt (Belgium). Each item of PAQ-A and the total PAQ-A score were correlated with the peak oxygen uptake, i.e. VOnd 40 W) . These lScoring of PAQs was performed as described by Kowalski et al. . PAQ-C qPAQ-A questionnaire has been originally designed for adolescents aged 14 to 20. The PAQ-A questionnaire consists of only nine questions  but it has the same scoring method as PAQ-C. The first question of both PAQs contained a checklist of 22 common leisure and sport activities as well as two ‘other’ fill-in choices. The first question was scored as the mean of all activities by a score from 1 to 5. The total score of these questionnaires was calculated by adding all questions’ average scores.s) corrected for age, gender, height and weight. Data were statistically analyzed using SPSS software version IBM 20.0 . Significance levels were set at P < 0.05.To determine the I-CVI, all six members of the expert committee rated each question/item of both PAQs in terms of its relevance to the underlying construct to a 4-point ordinal scale . Consequently, the I-CVI was computed for each item as the number of experts giving a rating of either 3 or 4 divided by the total number of experts. The S-CVI was calculated for each PAQ as the average of the I-CVIs for all items on the scale. An I-CVI higher than 0.78 was assumed to be excellent, and a minimum S-CVI of 0.80 was considered as acceptable . Inter-iTable The response rate of both PAQs ranged between 50 and 75%. In total, 196 PAQ-C and 95 PAQ-A were fully completed by both child and parent. Children (n = 5) who indicated they filled in the exact same answers as their parent were excluded from this analysis. Consequently, 192 PAQ-C questionnaires were completed by 101 girls and 91 boys aged between 5 and 12 (mean age: 8.9 ± 1.7 years), 94 PAQ-A questionnaires were completed by 52 girls and 42 boys aged between 12 and 17 (mean age: 13.6 ± 1.4 years)  and 0.758 (95% CI: 0.677-0.826), respectively.Table We experienced that PAQ-C was difficult to complete for 5 and 6-year-old children (n = 24) because of their limited reading skills. Therefore, we performed an additional analysis without the 5 and 6-year-old children, which showed a good overall reliability  and a better inter-rater reliability of total physical activity level  compared with the original analysis including this age group.2; mean age: 14.2 ± 1.8 years; 18 boys) and 16 normal-weight  adolescents completed the PAQ-A on the same day as the CPET took place. Descriptive characteristics of the study population are presented in Table 2 peak was 2201 ± 500 ml/min in obese and 2498 ± 835 ml/min in normal-weight individuals (P = 0.306). The total mean VO2 peak was 2309 ± 650 ml/min.A subset of 28 obese  PAQ-A items were not significantly associated with VO2 peak (P > 0.05). Overall, total PAQ-A score showed a moderate correlation with VO2 peak .PAQ-A is scored with an arbitrary numeric score, whereas the exercise monitor raw output is a scale count. The results presented in Table We tested whether both PAQs were still reliable in a small but independent cohort consisting of 47 subjects Table . These s2 peak was moderate.In this study, we evaluated the reliability and validity of a Dutch version of the PAQ-C and PAQ-A in a convenient sample of children and adolescents living in Flanders, Belgium. We report excellent content validity, an acceptable inter-item reliability and moderate to good strength of inter-rater agreement of PAQ-C (completed by children aged 5 to 12) and PAQ-A (completed by adolescents aged 12 to 17). In addition, the concurrent validity correlation of PAQ-A with the VOTo describe the level and pattern of physical activity, a standardized, reliable and valid instrument is essential. Furthermore, in children and adolescents it is important to use instruments which are non-invasive, easy-to-use and time-saving. To our knowledge, there are only a few validated short questionnaires in Dutch to assess the overall level of physical activity in adolescents , 14 and In our study, adolescents generally showed lower levels of physical activity compared to children. Janz et al.  also fouContent validity of the Dutch PAQ-C and PAQ-A was evaluated by the expert committee and was found to be excellent. Questions concerning sport activities in spare time, after-school, in the evening and the weekend were found highly relevant, whereas questions concerning break and lunchtime were scored lowest. This finding can be explained by the fact that break and lunchtime activities at school are relatively short in time and depend on whether the school itself organizes sport activities during break or lunch. Sport activities in spare time, after-school, in the evening or weekend are often practiced in sports associations and last longer in time. Nonetheless, we can conclude that the content of both PAQs is very relevant.PAQ-C and PAQ-A showed an acceptable inter-item reliability, which is in line with previous reports , 17, 18.Because of the short time frame of our study and from practical point of view we opted to study inter-rater instead of test-retest reliability. We found a moderate to good strength of inter-rater agreement. Accordingly, we conclude that these questionnaires are reliable for their use in the assessment of the overall level of physical activity in children and adolescents speaking Dutch. However, some marginal notes have to be made. Firstly, both PAQs can also be implemented in other Dutch speaking countries provided that the sport activities practiced in the respective country are used in the questionnaires. Nevertheless, caution has to be taken since Moore et al.  proved t2 peak was moderate. However, it is considerable higher than the previously reported associations between PAQ-A and accelerometry [The concurrent validity correlation between the Dutch PAQ-A total score and the VOerometry , 8, 17. This is the first validation study of a Dutch version of the PAQ-C and PAQ-A questionnaires for children and adolescents. Our results show that the Dutch versions of both PAQ-C and PAQ-A, provide reliable and valid estimates of physical activity among 5 to 17-year-old children and adolescents. Both questionnaires can be considered as very useful in clinical practice to assess overall level of physical activity in children and adolescents. Eventually, assessment of physical activity both at individual and at population level, could lead to improvement of personalized interventions and new school policies in order to prevent as well as to combat weight gain and associated health complications.Additional file 1: Dutch Physical Activity Questionnaire for Children (PAQ-C).(PDF 175 KB)Additional file 2: Dutch Physical Activity Questionnaire for Adolescents (PAQ-A).(PDF 118 KB)